Method and system for characterization and mapping of tissue lesions

ABSTRACT

The present invention provides a method and an apparatus for the in vivo, non-invasive, early detection of alterations and mapping of the grade of these alterations, caused in the biochemical and/or in the functional characteristics of epithelial tissues during the development of tissue atypias, dysplasias, neoplasias and cancers. The method is based, at least in part, on the simultaneous measurement of the spatial, temporal and spectral alterations in the characteristics of the light that is re-emitted from the tissue under examination, as a result of a combined tissue excitation with light and special chemical agents. The topical or systematic administration of these agents result in an evanescent contrast enhancement between normal and abnormal areas of tissue. The apparatus enables the capturing of temporally successive imaging in one or more spectral bands simultaneously. Based on the measured data, the characteristic curves that express the agent-tissue interaction kinetics, as well as numerical parameters derived from these data, are determined in any spatial point of the examined area. Mapping and characterization of the lesion, are based on these parameters.

RELATED APPLICATIONS

[0001] This application claims priority to Greek National ApplicationSer. No. 20000100102, filed on Mar. 28, 2000 and to United StatesNational application Ser. No. 09/739089 filed on Dec. 15, 2000.

FIELD OF THE INVENTION

[0002] The present invention is directed to a method and apparatus forthe in vivo, non-invasive detection and mapping of the biochemicaland/or functional pathologic alterations of human tissues.

BACKGROUND OF THE INVENTION

[0003] Cancer precursor signs are the so-called pre-cancerous states,which are often curable if they are detected at an early stage. If leftuntreated, the pre-cancerous state can develop into invasive cancer,which can subsequently metastasize. At this stage, the possibilities ofsuccessful therapy are dramatically diminished. Consequently, the earlydetection and the objective identification of the severity of thepre-cancerous state are of crucial importance.

[0004] Conventional methods that utilize optical instruments are verylimited in their ability to detect cancerous and pre-cancerous tissuelesions. This is due to the fact that the structural and metabolicchanges, which take place during the development of the disease, do notsignificantly and specifically alter the spectral characteristics of thepathological tissue.

[0005] In order to obtain a more accurate diagnosis, biopsy samples areobtained from suspicious areas, which are submitted for histologicalexamination. However, biopsies pose several problems, such as a) a riskfor sampling errors associated with the visual limitations in detectingand localizing suspicious areas; b) a biopsy can alter the naturalhistory of the intraepithelial lesion; c) mapping and monitoring of thelesion require multiple tissue sampling, which is subjected to severalrisks and limitations; and d) the diagnostic procedure performed withbiopsy sampling and histologic evaluation is qualitative, subjective,time consuming, costly and labor intensive.

[0006] In recent years, a few methods and systems have been developed toovercome the disadvantages of the conventional diagnostic procedures.These methods can be classified into two categories: a) methods whichare based on the spectral analysis of tissues in vivo, in an attempt toimprove the diagnostic information, and b) methods which are based onthe chemical excitation of tissues with the aid of special agents, whichcan interact with pathologic tissue and alter its opticalcharacteristics selectively, thus enhancing the contrast between lesionand healthy tissue.

[0007] In the first case, the experimental use of spectroscopictechniques has been motivated by the ability of these techniques todetect alterations in the biochemical and/or the structuralcharacteristics of tissue as the disease progresses. In particular,fluorescence spectroscopy has been extensively used in various tissuesWith the aid of a light source (usually laser) of short wave length(blue-ultraviolet range), the tissue is first excited. Next, theintensity of the fluorescent light emitted by the tissue as a functionof the wavelength of the light is measured.

[0008] Garfield and Glassman in U.S. Pat. Nos. 5,450,857 and Ramanajumet al. in 5,421,339 have presented a method based on the use offluorescence spectroscopy for the diagnosis of cancerous andpre-cancerous lesions of the cervix. The main disadvantage offluorescence spectroscopy is that the existing biochemical modificationsassociated with the progress of the disease are not manifested in adirect way as modifications in the measured fluorescence spectra. Thefluorescence spectra contain limited diagnostic information for twobasic reasons: a) Tissues contain non-fluorescent chromophores, such ashemoglobin. Absorption by such chromophores of the emitted light fromfluorophores can result in artificial dips and peaks in the fluorescencespectra. In other words the spectra carry convoluted information forseveral components and therefore it is difficult assess alterations intissue features of diagnostic importance; and b) The spectra are broadbecause a large number of tissue components are optically excited andcontribute to the measured optical signal. As a result, the spectra donot carry specific information of the pathologic alterations and thusthey are of limited diagnostic value. In short, the aforementionedfluorescent technique suffers from low sensitivity and specificity inthe detection and classification of tissue lesions.

[0009] Aiming to enhance the sensitivity and specificity of thepreceding method, Ramanujan et al. in the Patent No. WO 98/24369 havepresented a method based on the use of neural networks for the analysisof the spectral data. This method is based on the training of acomputing system with a large number of spectral patterns, which havebeen taken from normal and from pathologic tissues. The spectrum that ismeasured each time is compared with the stored spectral data,facilitating in this way the identification of the tissue pathology.

[0010] R. R. Kortun et al, in U.S. Pat. No. 5,697,373, seeking toimprove the quality of the measured diagnostic information, havepresented a method based on the combination of fluorescence spectroscopyand Raman scattering. The latter has the ability of providing moreanalytical information; however, Raman spectroscopy requires complexinstrumentation and ideal experimental conditions, which substantiallyhinders the clinical use thereof.

[0011] It is generally known that tissues are characterized by the lackof spatial homogeneity. Consequently the spectral analysis ofdistributed spatial points is insufficient for the characterization oftheir status.

[0012] Dombrowski in U.S. Pat. No. 5,424,543, describes amulti-wavelength, imaging system, capable of capturing tissue images inseveral spectral bands. With the aid of such a system it is possible ingeneral to map characteristics of diagnostic importance based on theirparticular spectral characteristics. However, due to the insignificanceof the spectral differences between normal and pathologic tissue, whichis in general the case, inspection in narrow spectral bands does notallow the highlighting of these characteristics and even more so, theidentification and staging of the pathologic area.

[0013] D. R. Sandison et al., in U.S. Pat. No. 5,920,399, describe animaging system, developed for the in vivo investigation of cells, whichcombines multi-band imaging and light excitation of the tissue. Thesystem also employs a dual fiber optic bundle for transmitting lightfrom the source to the tissue, and then from the tissue to an opticaldetector. These bundles are placed in contact with the tissue, andvarious wavelengths of excitation and imaging are combined in attempt toenhance the spectral differentiation between normal and pathologictissue.

[0014] In U.S. Pat. No. 5,921,926, J. R. Delfyett et al. have presenteda method for the diagnosis of diseases of the cervix, which is based onthe combination of Spectral Interferometry and Optical. CoherenceTomography (OCT). This system combines three-dimensional imaging andspectral analysis of the tissue.

[0015] Moreover, several improved versions of colposcopes have beenpresented, (D. R. Craine et al., U.S. Pat. Nos. 5,791,346 and K. L.Blaiz 5,989,184) in most of which, electronic imaging systems have beenintegrated for image capturing, analysis of tissue images, including thequantitative assessment of lesion's size. For the enhancement of theoptical differentiation between normal and pathologic tissue, specialagents are used in various fields of biomedical diagnostics, which areadministered topically or systematically. Such agents include aceticacid solution, toluidine blue, and various photosensitizers(porphyrines) (S. Anderson Engels, C. Klinteberg, K. Svanberg, S.Svanberg, In vivo fluorescence imaging for tissue diagnostics, Phys Med.Biol. 42 (1997) 815-24). The selective staining of the pathologic tissuearises from the property of these agents to interact with the alteredmetabolic and structural characteristics of the pathologic area. Thisinteraction enhances progressively and reversibly the differences in thespectral characteristics of reflection and/or fluorescence betweennormal and pathologic tissue. Despite the fact that the selectivestaining of the pathologic tissue is a dynamic phenomenon, in clinicalpractice the intensity and the extent of the staining are assessedqualitatively and statically. Furthermore, in several cases of earlypathologic conditions, the phenomenon of temporary staining afteradministering the agent, is short-lasting and thus the examiner is notable to detect the alterations and even more so, to assess theirintensity and extent. In other cases, the staining of the tissueprogresses very slowly, resulting in patient discomfort and the creationof problems for the examiner in assessing the intensity and extent ofthe alterations, since they are continuously changing. The above have asdirect consequence the downgrading of the diagnostic value of thesediagnostic procedures. Thus, their usefulness is limited to facilitatingthe localization of suspected areas for obtaining biopsy samples.

[0016] Summarizing the above, the following conclusions are drawn:

[0017] a) Various conventional light dispersion spectroscopic techniques(fluorescence, elastic, non-elastic scattering, etc.) have been proposedand experimentally used for the in vivo detection of alterations in thestructural characteristics of pathologic tissue. The main disadvantageof these techniques is that they provide point information, which isinadequate for the analysis of the spatially non-homogenous tissue.Multi-band imaging-has the potential to solve this problem by providingspectral information, of lesser resolution as a rule, in any spatialpoint of the area under examination. These imaging and non-imagingtechniques, however, provide information of limited diagnostic valuebecause the structural tissue alterations, which accompany thedevelopment of the disease, are not manifested as significant andcharacteristic alterations in the measured spectra. Consequently, thecaptured spectral information cannot be directly correlated with thetissue pathology, a fact that limits the clinical usefulness of thesetechniques.

[0018] b) The conventional (non-spectral) imaging techniques provide thecapability of mapping characteristics of diagnostic importance in two orthree dimensions. They are basically used for measuring morphologicalcharacteristics and as clinical documentation tools.

[0019] c) The diagnostic methods that are based on the selectivestaining of pathologic tissue with special agents allow the enhancementof the optical contrast between normal and pathologic tissue.Nevertheless they provide limited information for the in vivoidentification and staging of the disease.

[0020] The selective interaction of pathologic tissue with the agents,which enhance the optical contrast with healthy tissue, is a dynamicphenomenon. It is therefore reasonable to suggest that the measurementand analysis of kinetic properties could provide important informationfor the in vivo detection, identification and staging of tissue lesions.In a previous publication, in which one of the inventors is a co-author,(C. Balas, A. Dimoka, E. Orfanoudaki, E. koumandakis, “In vivoassessment of acetic acid-cervical tissue interaction using quantitativeimaging of back-scattered light: Its potential use for the in vivocervical cancer detection grading and mapping”, SPIE-Optical Biopsiesand Microscopic Techniques, Vol. 3568 pp. 31-37, (1998)), measurementsof the alterations in the characteristics of the back-scattered light asa function of wave-length and time are presented. These alterationsoccur in the cervix by the topical administration of acetic acidsolution. In this particular case, a general-purpose multi-spectralimaging system built around a tunable liquid crystal monochromator wasused for measuring the variations in intensity of the back-scatteredlight as a function of time and wavelength at selected spatial points.It was found that the lineshapes of curves of intensity ofback-scattered light versus time provide advanced information for thedirect identification and staging of tissue neoplasias. Unpublishedresults of the same research team indicate that similar results can alsobe obtained with other agents, which have the property of enhancing theoptical contrast between normal and pathologic tissue. Nevertheless, theexperimental method employed in the published paper is characterized byquite a few disadvantages, such as: The imaging monochromator requirestime for changing the imaging wavelength and as a consequence it isinappropriate for multispectral imaging and analysis of dynamicphenomena. It does not constitute a method for the mapping of the gradeof the tissue lesions, as the presented curves illustrate the temporalalterations of intensity of the back-scattered light in selected points.The lack of data modeling and parametric analysis of kinetics data inany spatial point of the area of interest restricts the usefulness ofthe method in experimental studies and hinders its clinicalimplementation. The optics used for the imaging of the area of interestis of general purpose and does not comply with the special technicalrequirements for the clinical implementation of the method. Clinicalimplementation of the presented system is also hindered by the fact thatit does not integrate appropriate means for ensuring the stability ofthe relative position between the tissue surface and image capturingmodule during the snapshot imaging procedure. This is very importantsince small movements of the patient (i.e. breathing) are always presentduring the examination procedure. If, after the application of theagent, micro-movements occur while an image is being recorded, then thespatial features of the captured images may not be accurate. This maysubstantially reduce the accuracy of the calculation of the curves inany spatial point that express the kinetics of marker-tissueinteraction.

SUMMARY OF THE INVENTION

[0021] The present invention provides a method for monitoring theeffects of a pathology-differentiating agent on a tissue sample. Themethod includes applying a pathology differentiating agent, e.g., aceticacid, on a tissue sample and measuring a spectral property, such as anemission spectrum, of the tissue sample over time, thereby monitoringthe effects of a pathology differentiating agent on a tissue sample. Thetissue may be a sample from: the cervix of the uterus, the vagina, theskin, the uterus, the gastrointestinal track or the respiratory track.Without intending to be limited by theory, it is believed that thepathology-differentiating agent induces transient alterations in thelight scattering properties of the tissue, e.g., the abnormalepithelium.

[0022] In another aspect, the present invention features a method forthe in vivo diagnosis of a tissue abnormality, e.g., a tissue atypia, atissue dysplasia, a tissue neoplasia (such as a cervical intraepithelialneoplasia, CINI, CINII, CINIII) condylomas or cancer, in a subject. Themethod includes applying a pathology differentiating agent, e.g., anacetic acid solution or a combination of solutions selected from aplurality of acidic and basic solutions, to a tissue. The method furtherincludes exposing the tissue in the subject to optical radiation, andmonitoring the intensity of light emitted from the tissue over time,thereby diagnosing a tissue abnormality in a subject. The opticalradiation may be broad band optical radiation, preferably polarizedoptical radiation.

[0023] The non-invasive methods of the present invention are useful forin vivo early detection of tissue abnormalities/alterations. The methodsare also useful for mapping the grade of abnormalities/alterations inepithelial tissues during the development of tissue atypias, dysplasias,neoplasias and cancers.

[0024] In one embodiment, the tissue area of interest is illuminatedwith a broad band optical radiation and contacted with a pathologydifferentiating agent, e.g., an agent or a combination of agents whichinteract with pathologic tissue areas characterized by an alteredbiochemical composition and/or cellular functionality and provoke atransient alteration in the characteristics of the light that isre-emitted from the tissue. The light that is re-emitted from the tissuemay be in the form of reflection, diffuse scattering, fluorescence orcombinations or subcombinations thereof. The intensity of the lightemitted from the tissue may be measured, e.g., simultaneously, in everyspatial point of the tissue area of interest, at a given time point orover time (e.g., for the duration of agent-tissue interaction). Adiagnosis may be made based on the quantitative assessment of thespatial distribution of alterations in the characteristics of the lightre-emitted from the tissue at given time points before and after theoptical and chemical excitation of the tissue. The diagnosis may also bemade based on the spatial distribution of parameters calculated fromkinetics curves obtained from the light re-emitted from the tissue.These curves are simultaneously measured in every spatial point of thearea under examination during the optical and chemical excitation of thetissue.

[0025] In one embodiment of the invention, the step of tissueillumination comprises exposing the tissue area under analysis tooptical radiation of narrower spectral width than the spectral width ofthe light emitted by the illumination source. In another embodiment, thestep of measuring the intensity of light comprises measuring theintensity of the re-emitted light in a spectral band, the spectral widthof which is narrower than the spectral width of the detector'ssensitivity. In yet another embodiment, the step of measuring theintensity of light comprises measuring simultaneously the intensity ofthe re-emitted light in a plurality of spectral bands, the spectralwidths of which are narrower than the spectral width of the detector'ssensitivity.

[0026] In yet another aspect, the present invention features anapparatus for the in vivo, non-invasive early detection of tissueabnormalities/alterations and mapping of the grade of these tissueabnormalities/alterations caused in the biochemical and/or in thefunctional characteristics of epithelial tissues, during the developmentof tissue atypias, dysplasias, neoplasias and cancers. The apparatusincludes optics for collecting the light re-emitted by the area underanalysis, selecting magnification and focusing the image of the area.The apparatus may also include optical imaging detector(s), means forthe modulation, transfer, display and capturing of the image of thetissue area of interest. In addition, the apparatus can include acomputer, which has data storage, processing and analysis means, amonitor for displaying images, curves and numerical data, optics for theoptical multiplication of the image of the tissue area of interest, anda light source for illuminating the area of interest. The apparatus mayalso include optical filters for selecting the spectral band of imagingand illumination, means for transmitting light and illuminating the areaof interest, control electronics, and optionally, software for theanalysis and processing of data. The software can help with the tissueimage capturing and storing in specific time points and for a pluralityof time points, before and after administration of thepathology-differentiating agent.

[0027] Using the foregoing apparatus, an image or a series of images maybe created which express the spatial distribution of the characteristicsof the kinetics of the induced alterations in the tissue's opticalcharacteristics, before and after the administration of the agent. Pixelvalues of the image correspond to the spatial distribution of thealterations in the intensity of the light emitted from the tissue atgiven times, before and after the optical and chemical excitation oftissue. The spatial distribution of parameters may be associated withpixel gray values as a function of time. The foregoing function may becalculated from the measured and stored images and for each row ofpixels with the same spatial coordinates.

[0028] In one embodiment, the step of optical filtering the imagingdetector comprises an optical filter that is placed in the optical pathof the rays that form the image of the tissue, for the recording oftemporally successive images in a selected spectral band, the spectralwidth of which is narrower than the spectral width of the detector'ssensitivity.

[0029] In yet another embodiment, the image multiplication opticsincludes light beam splitting optics that creates two identical imagesof the area of interest. The images are recorded by two imagingdetectors, in front of which optical filters are placed. The filters arecapable of transmitting light having a spectral width that is shorterthan the spectral width of the detector's sensitivity, so that twogroups of temporally successive images of the same tissue area arerecorded simultaneously, each one corresponding to a different spectralband.

[0030] In another embodiment, the image multiplication optics includemore than one beam splitter for the creation of multiple identicalimages of the area of interest. The images are recorded by multipleimaging detectors, in front of which optical filters are placed. Thefilters have different transmission characteristics and are capable oftransmitting light of spectral width shorter than the spectral width ofthe detector's sensitivity. Thus, multiple groups of temporallysuccessive images of the same tissue area are recorded simultaneously,each one corresponding to a different spectral band.

[0031] In a further embodiment, the image multiplication optics compriseone beam splitter for the creation of multiple identical images of thearea of interest, which are recorded by multiple imaging detectors, infront of which optical filters are placed with, preferably, differenttransmission characteristics and capable of transmitting light ofspectral width shorter than the spectral width of the detector'ssensitivity, so that multiple groups of temporally successive images ofthe same tissue area are recorded simultaneously, each one correspondingto a different spectral band.

[0032] In yet a further embodiment, the image multiplication opticsinclude one beam splitter for the creation of multiple identical imagesof the area of interest, which are recorded in different sub-areas ofthe same detector. Optical filters having different transmissioncharacteristics are placed in the path of the split beams. The filtersare capable of transmitting light of spectral width shorter than thespectral width of the detector's sensitivity. Multiple groups oftemporally successive images of the same tissue area are recordedsimultaneously in the different areas of the detector, each onecorresponding to a different spectral band.

[0033] In another embodiment, the step of filtering the light sourcecomprises an optical filter, which is placed in the optical path of anillumination light beam, and transmits light of spectral width shorterthan the spectral width of sensitivity of the detector used.

[0034] In a further embodiment, the step of filtering the light sourceincludes providing a plurality of optical filters and a mechanism forselecting the filter that is disposed in the path of the illuminationlight, thus enabling the tuning of the center wavelength and thespectral width of the light illuminating the tissue.

[0035] In another embodiment, the mapping of the grade of thealterations associated with the biochemical and/or functionalcharacteristics of the tissue area of interest is based on the pixelvalues of one image from the group of the recorded temporally successiveimages of the tissue area of interest.

[0036] In a further embodiment, this mapping is based on the pixelvalues belonging to a plurality of images, which are members of thegroup of the recorded temporally successive images of the tissue area ofinterest.

[0037] In another embodiment, this mapping is based on numerical dataderived from the pixel values belonging to a plurality of images, whichare members of the group of the recorded temporally successive images ofthe tissue area of interest.

[0038] In a further embodiment, a pseudo-color scale, which representswith different colors the different pixel values of the image or of theimages used for the mapping of abnormal tissue areas, is used for thevisualization of the mapping.

[0039] In one embodiment, the image or images are used for the in vivodetection, and identification of the borders of epithelial lesions.

[0040] In another embodiment, the pixel values of the image or of theimages, which are determined for the mapping of the grade of alterationsin biochemical and/or functional characteristics of tissue, are used asdiagnostic indices for the in vivo identification and staging ofepithelial lesions.

[0041] In yet another embodiment, the image or the images can besuperimposed on the color or black and white image of the same area oftissue under examination displayed on the monitor. Abnormal tissue areasare highlighted and their borders are demarcated, facilitating theselection of a representative area for taking a biopsy sample, theselective surgical removal of the abnormal area and the evaluation ofthe accuracy in selecting and removing the appropriate section of thetissue.

[0042] In a further embodiment, the image or the images which aredetermined for the mapping of the grade of alterations in biochemicaland/or functional characteristics of tissue are used for the evaluationof the effectiveness of various therapeutic modalities such asradiotherapy, nuclear medicine treatments, pharmacological therapy, andchemotherapy.

[0043] In another embodiment, the optics for collecting the lightre-emitted by the area under analysis includes optomechanical componentsemployed in microscopes used in clinical diagnostic examinations,surgical microscopes, colposcopes and endoscopes.

[0044] In one embodiment of the invention directed to colposcopyapplications, the apparatus may comprise a speculum, an articulated armonto which the optical head is attached. The optical head includes arefractive objective lens, focusing optics, a mechanism for selectingthe magnification, an eyepiece, a mount for attaching a camera, and anilluminator. The speculum is attached so that the central longitudinalaxis of the speculum is perpendicular to the central area of theobjective lens. Thus, when the speculum is inserted into the vagina andfixed in it, the relative position of the image-capturing optics and ofthe tissue area of interest remain unaltered, regardless ofmicro-movements of the cervix, which are taking place during theexamination of the female subject.

[0045] In a further embodiment, the apparatus may further comprise anatomizer for delivering the agent. The atomizer is attached to thearticulated arm-optical head of the apparatus and in front of thevaginal opening, where the spraying of the tissue may be controlled andsynchronized with a temporally successive image capturing procedure withthe aid of electronic control means.

[0046] In another embodiment of the apparatus of the invention, theimage capturing detector means and image display means include a camerasystem. The camera system has a detector with a spatial resolutiongreater than 1000×1000 pixels and a monitor of

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1 is a schematic representation of the present method's basicprinciple. at least 17 inches/43.18 cm (diagonal), so that highmagnification is ensured together with a large field of view while theimage quality is maintained.

[0048] In a further embodiment directed to microscopes used in clinicaldiagnostic examinations surgical microscopes and colposcopes, a systemincludes an articulated arm onto which the optical hand is attached. Theoptical head includes an objective lens, focusing optics, a mechanismfor selecting the magnification, an eyepiece, a mount for attaching acamera, an illuminator and two linear polarizers. One linear polarizeris disposed in the obptical path of the illuminating light beam and theother in the optical path of the rays, that form the image of thetissue. The polarization planes of these polarizers may be rotated. Whenthe planes are perpendicular to each other, the contribution of thetissue's surface reflection to the formed image is eliminated.

[0049] In another embodiment directed to endoscopy, an endoscope mayinclude optical means for transferring light from the light source tothe tissue surface. The optical means may also allow the collection andtransferring of rays along substantially the same axis. The opticalmeans also al low the focusing of the rays that form the image of thetissue. The endoscope may also include two linear polarizers. One linearpolarizer is disposed in the optical path of the illuminating light beamand the other in the optical path of the rays that form the image of thetissue. The polarization planes of these polarizers may be rotated. Whenthe planes are perpendicular to each other, the contribution of thetissue's surface reflection to the formed image is eliminated.

[0050] In another embodiment, microscopes used in clinical diagnosticexaminations, surgical microscopes and colposcopes may include areflective objective lens that replaces a retroactive lens. Thereflective objective lens is contracted so that a second reflectionmirror is disposed in the central part of its optical front aperture. Inthe rear, non-reflective part of this mirror, illumination means areattached from which light is emitted toward the object. With or withoutillumination zooming and focusing optics, the central ray or the emittedlight cone is coaxial with the central ray of the light-beam that entersthe imaging lens. With the aid of illumination zooming and focusingoptics, which may be adjusted simultaneously and automatically with themechanism for varying the magnification the optical imaging system, theilluminated area and the field-of-view of the imaging system can varysimultaneously and proportionally. Any decrease in image brightnesscaused by increasing the magnification is compensated with thesimultaneously zooming and focusing of the illumination beam.

[0051] Other features and advantages of the invention will he apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 is a schematic representation of the present method's basicprinciple.

[0053] In FIG. 1, the tissue (T), is sprayed using an atomizer (A),which contains the agent, e.g., acetic acid. At the same time, thetissue is illuminated with a source that emits light having a frequencywithin a specific spectral band that depends on the opticalcharacteristics of both the agent and the tissue. The characteristics ofthe light emitted from the source can be controlled by choosingparticular sources (LS), and optical filters (OFS). Sources of light forilluminating the tissue include light emitting diodes, and lasers.

[0054] For imaging the area of interest, light collection optics (L) maybe used, which focus the image onto a two-dimensional optical detector(D). The output signal of the latter is amplified, modulated anddigitized with the aid of appropriate electronics (EIS) and finally theimage is displayed on a monitor (M) and stored in the data-storing meansof a personal computer (PC). Between tissue (T) and detector (D),optical filters (OFI) can be interposed. The filter can be interposedfor tissue (T) imaging in selected spectral bands, at which the maximumcontrast is obtained between areas that are subjected to different gradeof alterations in their optical characteristics after administering theappropriate agent.

[0055] Before administration of the latter, images can be obtained andused as references. After the agent has been administered, the detector(D) helps to capture images of the tissue, in successive time instances,which are then stored in the computer's data-storage means. Themeasuring rate is proportional to the rate at which the tissue's opticalcharacteristics are altered, following the administration of the agent.

[0056] As used herein, an optical property, P, is a property that arisesfrom the interaction of electromagnetic waves and a material sample,e.g., a tissue, such as a tissue within a subject. For example, theproperty can be the intensity of light after it interacts with matter,as manifested by an absorption, emission, or Raman spectrum. A dynamicoptical property is a property that is obtained from a time-dependentoptical property, P(t), and is determined from the measurement of P(t)at more than one time. For example, a dynamical optical property can bea relaxation time, or a time integral of P(t).

[0057] In FIG. 1, images of the same tissue area are schematicallyillustrated, which have been stored successively before and afteradministering the agent (STI). In these images, the black areasrepresent tissue areas that do not alter their optical characteristics(NAT), while the gray-white tones represent areas that alter theiroptical characteristics (AT), following the administration of the agent.The simultaneous capture of the intensity of the light re-emitted fromevery spatial point of the tissue area under analysis and inpredetermined time instances, allows the calculation of the kinetics ofthe induced alterations.

[0058] In FIG. 1, two curves are illustrated: pixel value at position xy(Pv_(xy)), versus time t. The curve ATC corresponds to an area whereagent administration induced alterations (AT) in the tissue's opticalcharacteristics. The curve (NATC) corresponds to an area where noalteration took place (NAT).

[0059] Each pixel, (x,y), can be associated with a pixel value, such asintensity I, which generally depends on time. For example, at time t_(i)and pixel (x,y), the pixel value can be denoted by PV_(xy)(t_(i)). Oneuseful dynamical spectral property, which can be obtained by measuringpixel value versus time at a particular pixel (x,y), is the relaxationtime t_(rel)(x,y). Letting the maximum of a Pv_(xy) versus time curve bedenoted by A, then t_(rel)(x,y) satisfies PV_(xy)(t_(rel))=A/e, where eis the base of the natural logarithm. For example, if the pixel valueversus time curve can be approximated by an exponential with relaxationrate r, PV_(XY)(t)=A exp(−rt), where r>0, then t_(rel)(x,y)=1/r.

[0060] The calculation of these parameters (P) at every spatial point ofthe area under analysis allows kinetic information (KI) to be obtained,with pixel values that are correlated with these parameters. Thesevalues can be represented with a scale of pseudocolors (P_(min),P_(max)), the spatial distribution of which allows for immediate opticalevaluation of the intensity and extent of the induced alterations.Depending on the correlation degree between the intensity and the extentof the induced alterations with the pathology and the stage of thetissue lesion, the measured quantitative data and the derived parametersallow the mapping, the characterization and the border-lining of thelesion. The pseudocolor image of the phenomenon's kinetics (KI), whichexpresses the spatial distribution of one or more parameters, can besuperimposed (after being calculated) on the tissue image, which isdisplayed in real-time on the monitor. Using the superimposed image as aguide facilitates the identification of the lesion's boundaries, forsuccessful surgical removal of the entire lesion, or for locatingsuspicious areas to obtain a biopsy sample(s). Furthermore, based on thecorrelation of the phenomenon's kinetics with the pathology of thetissue, the measured quantitative data and the parameters that derivefrom them can provide quantitative clinical indices for the in vivostaging of the lesion or of sub-areas of the latter.

[0061] In some cases it is necessary to capture the kinetics of thephenomenon in more than one spectral band. This can help in the in vivodetermination of illumination and/or imaging spectral bands at which themaximum diagnostic signal is obtained. Furthermore, the simultaneousimaging in more than one spectral band can assist in minimizing thecontribution of the unwanted endogenous scattering, fluorescence andreflection of the tissue, to the optical signal measured by thedetector. The measured optical signal comprises the optical signalgenerated by the marker-tissue interaction and the light emitted fromthe endogenous components of the tissue. In many cases, the recordedresponse of the components of the tissue constitutes noise since itoccludes the generated optical signal, which carries the diagnosticinformation. Therefore, separation of these signals, based on theirparticular spectral characteristics, results in the maximization of thesignal-to-noise ratio and consequently in the improvement of theobtained diagnostic information.

[0062]FIG. 2 illustrates a method for measuring in two spectral bandssimultaneously and in any spatial point of the area under analysis, thekinetics of the alterations in the characteristics of the light emittedfrom the tissue, before and the after the administration of the contrastenhancing agent. The light emitted from the tissue is collected andfocused by the optical imaging module (L) and allowed to pass through abeam splitting (BSP) optical element. Thus, two identical images of thetissue (T) are generated, which can be captured by two detectors (D1,D2). In front of the detector, appropriate optical filters (Of_(λ1)),(Of_(λ2)) can be placed, so that images with different spectralcharacteristics are captured. Besides beam splitters, optical filters,dichroic mirrors, etc., can also be used for splitting the image of theobject. The detectors (D1), (D2) are synchronized so that they capturesimultaneously the corresponding spectral images of the tissue(Ti)_(λ1)), (Ti_(λ2)) and in successive time-intervals, which are storedin the computer's data storage means. Generalizing, multiple spectralimages can be captured simultaneously by combining multiple splittingelements, filters and sources.

[0063]FIG. 3 illustrates another method for capturing in differentspectral bands simultaneously and in any spatial point of the area underanalysis, the kinetics of the alterations in the characteristics of thelight emitted from the tissue, before and the after the administrationof the contrast enhancing agent. With the aid of a special prism (MIP)and imaging optics, it is possible to form multiple copies of the sameimage onto the surface of the same detector (D). Various optical filters(OF_(λ1)), (OF_(λ2)), (OF_(λ3)), and (OF_(λ4)), can be interposed alongthe length of the optical path of the rays that form the copies of theobject's image, so that the multiple images correspond to differentspectral areas.

[0064] For the clinical use of the methods of the invention, thedifferent implementations of imaging described above can be integratedto conventional optical imaging diagnostic devises. Such devises are thevarious medical microscopes, colposcopes and endoscopes, which areroutinely used for the in vivo diagnostic inspection of tissues. Imagingof internal tissues of the human body requires in most cases theillumination and imaging rays to travel along the same optical path,through the cavities of the body. As a result, in the common opticaldiagnostic devices the tissue's surface reflection contributessubstantially to the formed image. This limits the imaging informationfor the subsurface characteristics, which is in general of greatdiagnostic importance. This problem becomes especially serious inepithelial tissues such as the cervix, larynx, and oral cavity, whichare covered by fluids such as mucus and saliva. Surface reflection alsoobstructs the detection and the measurement of the alterations in thetissue's optical properties, induced after the administration of agents,which enhance the optical contrast between normal and pathologic tissue.More specifically, when an agent alters selectively the scatteringcharacteristics of the pathologic tissue, the strong surface reflectionthat takes place in both pathologic (agent responsive) and normal (agentnon responsive) tissue areas, occludes the diagnostic signal thatoriginates from the interaction of the agent with the subsurfacefeatures of the tissue. In other words, surface reflection constitutesoptical noise in the diagnostic signal degrading substantially theperceived contrast between agent responsive and agent non-responsivetissue areas.

[0065] For accurate diagnoses using the aforementioned imaging devices,appropriate optics can be used to eliminate noise arising from surfacereflection. FIG. 4 illustrates a schematic diagram of a medicalmicroscope that includes a light source (LS), a magnification selectionmechanism (MS), an eyepiece (EP) and a mount for attaching the imagecapturing module (CA), (detector(s), readout electronics etc). Toeliminate surface reflection, a pair of linear polarizers is employed.Light from the source passes through a linear polarizer (LPO) with theresulting linearly polarized light (LS) then impinging on the tissue.The surface reflected light (TS) has the same polarization plane as theincident light (Fresnel reflection). By placing another linear polarizer(IPO), oriented at a right angle with respect to the first, in the pathof the light emitted from the tissue, the contribution of the surfacereflected light is eliminated. The light that is not surface reflectedenters the tissue, where due to multiple scattering, light polarizationis randomized. Thus, a portion of the re-emitted light passes throughthe imaging polarization optics, carrying improved information for thesubsurface features.

[0066]FIG. 5 illustrates an endoscope that includes an eyepiece (EP),which can be adapted to an electronic imaging system, and optical fibersor crystals for the transmission of both illumination and image rays.The endoscope also includes a first linear polarizer (LPO), disposed inthe optical path of the illumination rays (LE), and a second polarizer(IPO), oriented at right angles to the first, disposed in the path ofthe light emitted by the tissue (II). The polarizer (LPO) can bedisposed as shown in the figure, or, alternatively, where the lightenters the endoscope (IL). In the latter case, the endoscope has to beconstructed using polarization preserving crystals or fiber optics fortransferring the light. If polarization preserving light transmissionmedia are used, then the polarizers for the imaging rays can be disposedin their path, in front or in back of the eyepiece (EP).

[0067] A problem for the effective clinical implementation of the methoddescribed above involves the micro-movements of the patient, which arepresent during the snapshot imaging of the same tissue area. Thisproblem is eliminated when the patient is under anesthesia (opensurgery). In most cases, however, the movements of the tissue relativeto the image capturing module, occurring during the successive imagecapturing time-course, result in image pixels, with the same imagecoordinates, which do not correspond to exactly the same spatial pointx,y of the tissue area under examination. This problem is typicallyencountered in colposcopy. A method for eliminating the influence to themeasured temporal data of the relative movements between tissue andimage capturing module is presented below.

[0068] A colposcopic apparatus, illustrated in FIG. 6, includes anarticulated arm (AA), onto which the optical head (OH) is affixed. Thehead (OH) includes a light source (LS), an objective lens (OBJ), aneyepiece (EP) and optics for selecting the magnification (MS). Theimage-capturing module is attached to the optical head (OH), through anopto-mechanical adapter. A speculum (KD), which is used to open-up thevaginal canal for the visualization of the cervix, is connectedmechanically to the optical head (OH), so that its longitudinal symmetryaxis (LA) is perpendicular to the central area of the objective lens(OBJ). The speculum enters the vagina and its blades are opened upcompressing the side walls of the vagina. The speculum (KD), beingmechanically connected with the optical head (OH), transfers anymicromovement of the patient to the optical head (OH), which, beingmounted on an articulated arm (AA), follows these movements. Thus therelative position between tissue and optical head remains almostconstant.

[0069] An important issue that must also be addressed for the successfulclinical implementation of the diagnostic method described herein is thesynchronization of the application of the pathology differentiatingagent with the initiation of the snapshot imaging procedure. FIG. 6,illustrates an atomizer (A) attached to the optical head of themicroscope. The unit (MIC) is comprised of electronics for controllingthe agent sprayer and it can incorporate also the container for storingthe agent. When the unit (MIC) receives the proper command from thecomputer, it sprays a predetermined amount of the agent onto the tissuesurface, while the same or another command initiates the snapshot imagecapturing procedure.

[0070] The diagnostic examination of non-directly accessible tissueslocated in cavities of the human body (ear, cervix, oral cavity,esophagus, colon, stomach) is performed with the aid of common clinicalmicroscopes. In these devices, the illumination-imaging rays are nearco-axial. More specifically, the line perpendicular to the exit point oflight into the air, and the line perpendicular to the objective lens,form an angle of a few degrees. As a result, these microscopes operateat a specific distance from the subject (working distance), where theilluminated tissue area coincides with the field-of-view of the imagingsystem. These microscopes are found to be inappropriate in cases wheretissue imaging through human body cavities of small diameter and atshort working distances is required. These technical limitations hinderthe successful clinical implementation of the method described herein.As discussed above, elimination of surface reflection results in asubstantial improvement of the diagnostic information obtained from thequantitative assessment of marker-tissue interaction kinetics. If acommon clinical microscope is employed as the optical imaging module,then as a result of the above-mentioned illumination-imaging geometry,multiple reflections occur in the walls of the cavity before the lightreaches the tissue under analysis. Multiple reflections are morenumerous in colposcopy because of the highly reflective blades of thespeculum, which is inserted into the vagina to facilitate the inspectionof the cervix.

[0071] If the illuminator of the imaging apparatus emits linearlypolarized light, the multiple reflections randomize the polarizationplane of the incident light. As discussed above, if the light impingingon the tissue is not linearly polarized, then the elimination of thecontribution from the surface reflection to the image can not beeffective.

[0072]FIG. 7 illustrates an optical imaging apparatus that includes alight source located at the central part of its front-aperture. Withthis arrangement, the central ray of the emitted light cone is coaxialwith the central ray of the light beam that enters the imagingapparatus. This enables illumination rays to directly reach the tissuesurface under examination before multiple reflections occur with thewall of the cavity or speculum. A reflective-objective lens is used,which includes a first reflection (1RM) and a second reflection (2RM)mirror. A light source (LS) is disposed at the rear of the secondreflection mirror (2RM), together with, if required, optics for lightbeam manipulation such as zooming and focusing (SO). Thereflective-objective lens (RO), by replacing the commonrefractive-objective used in conventional microscopes, provides imagingcapability in cavities of small diameter with the freedom of choosingthe working distance. The zooming and focusing optics of the light beamcan be adjusted simultaneously with the mechanism for varying themagnification of the optical imaging system so that the illuminationarea and the field-of-view of the imaging system vary simultaneously andproportionally. Thus, image brightness is preserved regardless of themagnification level of the lens. The imaging-illumination geometryembodied in this optical imaging apparatus, along with the light beammanipulation options, helps to eliminate the surface reflectioncontribution to the image and consequently helps to efficientlyimplement the method described herein.

[0073] Equivalents

[0074] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed:
 1. Use of a differentiating agent in a method formonitoring the effects of said pathology differentiating agent on atissue, comprising: administering a pathology differentiating agent tosaid tissue, exposing said tissue to broad band, continuous wave opticalradiation; and measuring a dynamic optical property of said tissue,after said tissue has interacted with said pathology differentiatingagent, wherein said dynamic optical property is obtained from at leasttwo values of a time-dependent optical property, thereby monitoring theeffects of a pathology differentiating agent on a tissue.
 2. Use of adifferentiating agent in a method for monitoring the effects of saidpathology differentiating agent on a tissue, comprising: administering apathology differentiating agent to said tissue; exposing said tissue tooptical radiation; and measuring a non-fluorescent dynamic opticalproperty of said tissue, after said tissue has interacted with saidpathology differentiating agent, wherein said dynamic optical propertyis obtained from at least two values of a time-dependent opticalproperty, thereby monitoring the effects of a pathology differentiatingagent on a tissue.
 3. Use of a differentiating agent in a method formonitoring the effects of said pathology differentiating agent on atissue, the method comprising: administering a pathology differentiatingagent to said tissue; exposing said tissue to optical radiation; andwhile exposing said tissue to optical radiation, measuring a dynamicoptical property of said tissue, after said tissue been interacted withsaid pathology differentiating agent, wherein said dynamic opticalproperty is obtained from at least two values of a time-dependentoptical property, thereby monitoring the effects of a pathologydifferentiating agent on a tissue.
 4. Use of a differentiating agent ina method for monitoring and mapping the effects of said pathologydifferentiating agent on a tissue, comprising: administering a pathologydifferentiating agent to said tissue; exposing said tissue to broadband, continuous wave optical radiation; and after said tissue hasinteracted with said pathology differentiating agent, measuring adynamic optical property at several locations of the tissue to produce aspatial map of the tissue, wherein said dynamic optical property isobtained from at least two values of a time-dependent optical property,thereby monitoring and mapping the effects of a pathologydifferentiating agent on a tissue.
 5. The use of any one of claims 1, 2,3, or 4, further comprising measuring an optical property of said tissuebefore said tissue has interacted with said pathology differentiatingagent.
 6. The use of any one of claims 1, 2, 3, or 4, wherein saidpathology differentiating agent is selected from the group consisting ofan acidic solution and a basic solution.
 7. The use of any one of claims1, 2, 3, or 4, wherein said tissue is a tissue from the cervix of theuterus.
 8. The use of any one of claims 1, 2, 3, or 4, wherein saidtissue is a vagina tissue.
 9. The use of any one of claims 1, 2, 3, or4, wherein said tissue is a uterus tissue.
 10. The use of any one ofclaims 1, 2, 3, or 4, wherein said tissue is selected from the groupconsisting of a skin tissue, an oral cavity tissue, a gastrointestinaltrack tissue, and a respiratory track tissue.
 11. The use of adifferentiating agent in a method for the in vivo diagnosis and mappingof a tissue abnormality in a subject, comprising administering apathology differentiating agent to a subject; exposing a tissue in saidsubject to optical radiation; and while exposing said tissue to opticalradiation, monitoring the dynamic optical property of said tissue aftersaid tissue has interacted with said pathology differentiating agent,wherein said dynamic optical property is obtained from at least twovalues of a time-dependent optical property, thereby diagnosing andmapping a tissue abnormality in a subject.
 12. The use of claim 11,wherein said optical radiation is broad band optical radiation.
 13. Theuse of claim 11, wherein said optical radiation is polarized opticalradiation.
 14. The use of claim 11, wherein said tissue abnormality isselected from the group consisting of a tissue atypia, a tissuedysplasia, a tissue neoplasia, condylomas and cancer.
 15. The use ofclaim 11, wherein said tissue abnormality is neoplasia of any grade. 16.The use of claim 11, wherein said tissue abnormality is a cervicalintraepithelial neoplasia.
 17. The use of claim 11, wherein saidpathology differentiating agent is selected from the group consisting ofan acidic solution and a basic solution.
 18. The use of claim 11,wherein said tissue is a sample from the cervix of the uterus.
 19. Theuse of claim 11, wherein said tissue is a vaginal tissue.
 20. The use ofclaim 11, wherein said tissue is a uterine tissue.
 21. The use of claim11, wherein said tissue is selected from the group consisting of a skintissue, an oral cavity tissue a gastrointestinal track tissue, and arespiratory track tissue.
 22. A system for diagnosing and mapping atissue abnormality, the system comprising a detector for measuring adifference in a dynamic optical property between a normal tissue and anabnormal tissue, said difference being caused by applying a pathologydifferentiating agent to the tissue.
 23. The system of claim 22, furthercomprising an applicator for applying a pathology differentiating agentto the tissue.
 24. The system of claim 23, wherein the applicator is anatomizer.
 25. The system of claim 22, wherein the pathologydifferentiating agent is capable of altering the reflectioncharacteristics of the abnormal tissue.
 26. The system of claim 22,wherein the pathology differentiating agent is selected from the groupconsisting of an acidic solution and a basic solution.
 27. The system ofclaim 22, wherein the detector is suitable for obtaining an emissionspectrum for measuring the dynamic optical property of the tissue. 28.The system of claim 22, wherein the detector is suitable for measuringlight emitted by the tissue for measuring the dynamic optical propertyof the tissue.
 29. The system of claim 28, further comprising a beamsplitter for splitting the light emitted by the tissue into a pluralityof beams.
 30. The system of claim 29, further comprising a plurality ofoptical filters, each of the filters transmitting over a specificspectral range, for filtering each of the plurality of beams, whereinthe dynamic optical property of the tissue can be measured for each ofthe plurality of filtered beams.
 31. The system of claim 22, furthercomprising a light source for shining light on the tissue to obtain thedifference in the dynamic optical property.
 32. The system of claim 22,wherein the light source includes at least one of a laser and alight-emitting diode.
 33. The system of claim 22, wherein the lightshone on the tissue is linearly polarized in a first direction.
 34. Thesystem of claim 33, further comprising a linear polarizer for polarizinglight emitted from the tissue, in response to shining light thereon, ina direction substantially perpendicular to the first direction, therebyreducing a contribution of light reaching the detector arising fromtissue surface reflection.
 35. The system of claim 31, wherein the lightsource and the detector are included on an optical head.
 36. The systemof claim 35, further comprising a probing device connected to theoptical head, said probing device coupled with the tissue so thatrelative motion between the tissue and the optical head is substantiallyeliminated.
 37. The system of claim 36, wherein the tissue includesvaginal tissue, and the probing device includes a speculum for insertioninto a vagina.
 38. The system of claim 36, wherein the tissue includesgastrointestinal tissue or respiratory tissue, and the probing deviceincludes an endoscope for insertion into a gastrointestinal track or arespiratory track, respectively.
 39. The system of claim 36, furthercomprising a reflective objective lens so that the light source emits acone of light having a symmetry axis that is substantially coaxial witha longitudinal symmetry axis of the objective lens to substantiallyreduce multiple reflections of light by at least one of the probingdevice and the tissue.
 40. An apparatus for the in vivo diagnosis andmapping of a tissue abnormality comprising a. Optics for collectinglight re-emitted by the tissue under analysis for magnification andfocusing an image of the tissue; b. Optical imaging detector; c. Meansfor modulating, transferring, displaying and capturing of the image ofthe tissue; d. Computer which includes data storage, processing andanalysis means; e. Monitor for displaying images, curves and numericaldata; f. Optics for the optical multiplication of the image of thetissue; g. Light source consisting of broad band, continuous waveoptical radiation for illuminating the tissue; h. Optics filters orselecting a spectral band of imaging and illumination; i. Means fortransmitting light and illuminating the tissue; j. Control electronics;and k. Software for analyzing and processing of data, and for capturing,registering and storing of tissue images obtained at specific spectralbands and time instances, before and after administering an agent or acombination of agents that alter the optical properties of atypical andpathological tissue areas, wherein dynamic, optical properties and theirspatial map is obtained from the stored images.
 41. The apparatus ofclaim 40, wherein a pseudo-color scale, which represents differentvalues of the dynamic optical properties with different colors, is usedfor the visualization of tissue areas with abnormalities of differentgrade.
 42. The apparatus of claim 40 or 41, wherein the image is usedfor the selective surgical removal of the atypical and pathologicaltissue area.
 43. The apparatus of claim 40 or 41, wherein the image isused as a guide for taking a biopsy sample.
 44. The apparatus if claim40 or 41, wherein the image is used for the in vivo detection, mapping,and determination of the borders of epithelial lesions.
 45. Theapparatus or claim 40 or 41, wherein the parameters determined as afunction of dynamic optical properties are used as diagnostic indicesfor the in vivo identification, grading of epithelial lesions and forthe mapping of tissue areas with epithelial lesions of different grade.46. A system for diagnosing and mapping a tissue abnormality, the systemcomprising: a detector for measuring a difference in a dynamic opticalproperty between a normal tissue and an abnormal tissue, said differencebeing caused by applying a pathology differentiating agent to thetissue; a light source for shining light on the tissue, wherein thelight source and the detector are included on an optical head; a probingdevice connected to the optical head, said probing device coupled withthe tissue so that relative motion between the tissue and the opticalhead is substantially eliminated; and an applicator for applying apathology differentiating agent to the tissue.
 47. The system of claim46, wherein the tissue includes vaginal tissue, and the probing deviceincludes a speculum for insertion into a vagina.
 48. The system of claim46, wherein the tissue includes gastrointestinal tissue or respiratorytissue, and the probing device includes an endoscope for insertion intoa gastrointestinal track or a respiratory track, respectively.
 49. Thesystem of claim 46, further comprising a beam splitter for splitting thelight emitted by the tissue into a plurality of beams; a plurality ofoptical filters, each of the filters transmitting over a specificspectral range, for filtering each of the plurality of beams, whereinthe dynamic optical property of the tissue can be measured for each ofthe plurality of filtered beams.
 50. The system of claim 46, wherein thelight shone on the tissue is linearly polarized in a first direction.51. The system of claim 50, further comprising a linear polarizer forpolarizing light emitted from the tissue, in response to shining lightthereon, in a direction substantially perpendicular to the firstdirection, thereby reducing a contribution of light reaching thedetector arising from tissue surface reflection.